A novel gene delivery platform for single cell organisms

Microalgae are single-celled organisms that have great potential in producing biofuels from sunlight and carbon dioxide. In order to create an algal biofuel industrial process that is economically viable, the organisms must be genetically engineered so that they grow faster, consume fewer nutrients or require less light. A critical step in genetic engineering is delivering target DNA into the cell interior, but algae have a rigid cell wall that impedes this delivery. Current methods of genetic transformation are time-consuming and inefficient. To solve this problem, I am developing a promising new technology that increases the efficiency and the throughput of each transformation experiment. Briefly, this entails the application of hollow microneedle arrays that are loaded with DNA. The algae are pierced by these needles and the materials are injected into their cytoplasm for subsequent expression of target proteins or other molecules. In this presentation, I will highlight some recent progress of this project, which includes the preparation of the microneedle array platform and some preliminary results on algae piercing.

A novel gene delivery platform for single cell organisms

Microalgae are single-celled organisms that have great potential in producing biofuels from sunlight and carbon dioxide. In order to create an algal biofuel industrial process that is economically viable, the organisms must be genetically engineered so that they grow faster, consume fewer nutrients or require less light. A critical step in genetic engineering is delivering target DNA into the cell interior, but algae have a rigid cell wall that impedes this delivery. Current methods of genetic transformation are time-consuming and inefficient. To solve this problem, I am developing a promising new technology that increases the efficiency and the throughput of each transformation experiment. Briefly, this entails the application of hollow microneedle arrays that are loaded with DNA. The algae are pierced by these needles and the materials are injected into their cytoplasm for subsequent expression of target proteins or other molecules. In this presentation, I will highlight some recent progress of this project, which includes the preparation of the microneedle array platform and some preliminary results on algae piercing.

In order to prove the concept of gene delivery, we will be using a reporter gene such as orange fluorescent protein. Because this is an exogene, only the transfected cells will express it.

There are a few different methods of gene delivery currently used, including biolistic transformation, electroporation, and agitation with glass beads or whiskers. Each has its shortcomings: cost, low-throughput, specialized equipment, or removal of the algal cell wall. Our platform should overcome all these challenges.

Great presentation Andrew. Are there any technical challenges that need to be overcome in making the hollow needles? Also, do we know how the algae “heal” themselves once the genes are injected into the cytoplasm? Are there proof-of principle studies on this point?

I think that obtaining a uniformly thick gold deposition layer will be the critical challenge in obtaining hollow needles that have a narrow distribution in their tip openings. The thickness of the gold layer defines the relationship between the tip openings’ outer and inner diameters, which will need to be precisely tuned for optimal piercing of algae cells and transport of DNA through the openings.

Regarding the healing of the cells after piercing: this is not a concern for us because we do not intend for the pierced cells to leave the needles. Instead, the cells remain alive on the microneedle array and replicate according to normal cell function. The transfected genes are passed on to the daughter cells, which are not themselves pierced on the needles. If the transfection is successful, the new genes will remain with the entire culture from then on.

Thanks for your comment, Dr. Kong.
So far in our lab, we have not yet delivered any genes. However, during her post-doctoral research at the University of Florida, my advisor, Hitomi Mukaibo, was able to tether DNA to solid microneedles and pierce algae. She evaluated both cell viability and gene expression; the manuscript has been submitted to a journal for review.

Thank you, Dr. Noginova.
The shape of our needles is critical; the conical shape provides both a sharp point that is able to puncture the rigid cell wall as well as a wide base that provides mechanical strength. Because the microalgae have a cell wall, a significant force is required to pierce them, which sets our microneedle platform apart from others. Lastly, the template synthesis method we use is simple and does not require the nanofabrication techniques that many other nanoneedles are made with.

Thanks for commenting, Dr. Wei.
We have certainly thought about DNA concentration, but don’t yet have any numbers to reference. The amount of DNA required will clearly need to be optimized to provide a sufficiently high driving force for diffusion while minimizing cost.

Thanks for your comments, Myisha. The answer to your question is actually the focus of my poster presentation. The plug consists of the biopolymer chitosan and a crosslinker that contains a disulfide bond. The disulfide bond can be reduced, or cleaved, by free thiol-containing compounds within the algae cells. We highlight the tripeptide glutathione as a candidate reducing agent, and demonstrate its ability to degrade the plug in vitro.